Saliva maintains oral health. Building on our past studies of saliva formation and its alteration during pathology, we previously developed novel approaches to treat salivary dysfunction using principles of gene therapy, as well as strategies to use normal salivary glands as a gene transfer target site for treating systemic single protein deficiency disorders (SSPDDs). We have studied the value of transferring the human aquaporin-1 (hAQP1) cDNA to restore salivary flow in patients with irradiation (IR)-damaged salivary glands. Based on previous pre-clinical efficacy studies with a recombinant serotype 5 adenoviral (Ad5) vector encoding hAQP1 (AdhAQP1) conducted in rats and miniature pigs, and an extensive safety (toxicology and biodistribution) study of the AdhAQP1 vector in rats, we received an IND (BB-IND 13102) and approval to conduct a clinical trial for testing this vector in patients. The protocol, 06-D-0206, received all required NIH and external approvals. We began treating patients with this vector in 2008 and have delivered AdhAQP1 to eleven patients, i.e., completed the first three dose cohorts (3/cohort;4.8x10e7, 2.9x10e8 and 1.3x10e9 vector genomes, vg, to a single parotid gland), and treated two patients in the fourth dose cohort (5.8x10e9 vg/gland). We were unable to treat more patients, as the expiration date for the GMP vector was reached on May 10, 2011. All eleven treated patients tolerated the vector and study procedures well. There have been clear indications of efficacy, both subjective and objective, in five of the treated patients (one in the first dose cohort and two each in the second and third dose cohorts). Based on the results of this study, and preclinical efficacy with an AAV2 vector encoding hAQP1 in miniature pigs reported last year, it is likely that a clinical trial with the AAV2hAQP1 vector will soon be developed. We also used a pharmacologic approach to study if the stable nitroxide Tempol and D-methionine (D-met), were able to prevent oral mucositis in mice following exposure to IR +/- Cisplatin. Female C3H mice, 8 weeks old, were irradiated with five fractions (6-8 Gy) +/- Cisplatin to induce oral mucositis (lingual ulcers). Just prior to IR and chemotherapy, mice were treated, either alone or in combination, with different doses of Tempol and D-met. Significant lingual ulcers resulted from the 5 x 8 Gy IR fractions, which were enhanced with Cisplatin treatment. D-met provided stereospecific partial protection from lingual ulceration, while Tempol provided nearly complete protection. D-met plus a suboptimal Tempol dose also provided complete protection. Overall, these two fairly simple pharmacological treatments were able to markedly reduce chemoradiation-induced oral mucositis in mice. A key consideration for all clinical gene transfer applications is the absence of tight control of transgene expression. No approved clinical gene therapy trial has employed a vector that permits the regulation of transgene expression. During this past year we completed a GLP-level (FDA Good Laboratory Practice standards) safety study of an AAV2 vector encoding a rapamycin-responsive chimeric transcription factor that regulates the expression of a therapeutic transgene (human erythropoietin, hEpo). The vector, AAV2-TF2.3w-hEpo (2.5x10e7-2.5x10e10 vg), was administered to a single submandibular gland of male and female mice, and mediated hEpo expression in vivo following a rapamycin injection, but not in its absence. Control (saline-treated) and vector treated animals maintained their weight, and consumed food and water, similarly. Vector delivery led to no significant toxicological effects as judged by hematology, clinical chemistry, and gross and microscopic pathology evaluations. Overall, the results showed that AAV2-TF2.3w-hEpo delivery to salivary glands in mice was safe. As described in many past annual reports, we have shown in multiple animal models (mice, rats, miniature pigs and rhesus macaques) that salivary glands are a potentially useful gene transfer target site for treating certain SSPDDs. This year we have extended these studies with two novel potential clinical applications. Last year, we reported a possible way for salivary gland gene transfer to be used for treating diabetes mellitus (DM), using transfer of the cDNA for glucagon-like peptide 1. We have continued to explore possible salivary gene transfer strategies for treatment of DM testing an Ad5 vector encoding a naturally occurring human proinsulin variant (hproinsulin B10;Ad-proins-B10). An in vitro assay based on Akt/PKB phosphorylation at serine 473 following activation of the insulin receptor showed that the transgenic hproinsulin B10 secreted by Ad-proins-B10-transduced cells was bioactive. Ad-proins-B10 delivered to salivary glands of healthy male Balb/c mice by retroductal instillation yielded a dose-dependent increase in serum hproinsulin B10 at 24h post-transduction. This correlated with a decrease in blood glucose levels. Next, we used the beta-cell-specific cytotoxic drug, alloxan (ALX) to test the value of Ad-proins-B10 delivery to salivary glands as a treatment for ALX-induced Type I DM. After ALX treatment, mice were administered either Ad-proins-B10 or Ad-control (a control Ad5 vector), at 10e10 vg/gland, and serum glucose and hproinsulin B10 levels were monitored. Consistent with experiments in non-diabetic mice, serum hproinsulin B10 was significantly elevated in Ad-proins-B10-treated diabetic mice and these mice had blood glucose levels significantly reduced compared to Ad-control-treated mice. This demonstrates the potential therapeutic value of hproinsulin B10 gene delivery to salivary glands in Type I DM. All secreted proteins that we examined previously for applications with SSPDDs have been endocrine hormones with relatively easy to monitor biological activities in vivo, as with hproinsulin B10. Recently, we examined the expression and biochemical activity of transgenic human alpha-1-antitrypsin (hA1AT), a protease inhibitor whose biological activity is strictly conformation dependent, after production in rodent submandibular glands. An Ad5 vector (Ad.hA1AT) was constructed and first characterized in vitro. hA1AT expression was shown by ELISA and its biological activity demonstrated by the inhibition of human neutrophil elastase (hNE). Thereafter, Ad.hA1AT was administered to submandibular glands of rats and mice. The transgenic hA1AT expressed in submandibular glands was secreted primarily into the bloodstream, apparently correctly N-glycosylated, and inhibited hNE activity. Thus, after in vivo gene transfer, rodent salivary glands can produce a non-hormonal, transgenic, secretory glycoprotein exhibiting complex and conformation dependent biological activity.